Assembly of Artificial Oxidoreductases

蛋白质是由氨基酸的线性链构成的生物分子，这些氨基酸采用复杂的三维结构，这些结构由它们的氨基酸序列决定。每种蛋白质通常都有一个独特的结构，这种结构与它在自然界中执行的功能有着不可磨灭的联系。酶是催化细胞中发生的化学反应的蛋白质，例如，它有助于从呼吸作用和光合作用中捕获和储存化学能。新的人工蛋白质和酶的设计仍然是生物化学中的巨大挑战之一，考验着我们对蛋白质作为材料的本质的基本理解。解开天然酶发挥的极其强大的化学作用，有望为新的药物、疗法和可再生绿色能源的来源提供途径。大多数构建新酶的尝试都集中在对天然蛋白质和酶进行修饰以引入新的催化功能，并取得了一定程度的成功。与重新设计天然蛋白质相关的问题是由于自然界通过自然选择将其融入到蛋白质复杂的3D结构中的复杂性层次。这种复杂性使自然进化的蛋白质和酶的功能解构复杂化，使它们的重新设计变得极其困难。我们认为这种复杂性不是蛋白质和酶的必要特征。我们有效避免这种复杂性的方法是与自然选择未触及的蛋白质合作。这些简单的蛋白质，新蛋白质，是具有通用氨基酸序列的小而结实的蛋白质支架，可以作为模板，在其上添加天然蛋白质功能。某些蛋白质和酶的非蛋白质成分，如蛋白质血红蛋白的血红素分子，可以在新蛋白质中得到有效支持，这些分子在天然蛋白质中发挥的各种功能可以被开发出来。这种方法如何有效使用的一个例子是创造了一种能够可逆结合氧的血红素结合新蛋白，这是肌红蛋白、血红蛋白和最近发现的脑红蛋白的共同功能。工程学的功能元素是一步一步地添加的，形成这样一种蛋白质的要求在数量上令人惊讶地少。而且，由于大肠杆菌大量生产这种人造蛋白质，这种结合氧的新蛋白质的生产成本非常低，而且很容易通过标准的分子生物学技术进行改变。由于血红素蛋白质中的氧结合状态是天然蛋白质中许多催化过程的先决条件，我们计划从大自然中获得灵感，进一步将这些蛋白质开发成人造酶。我们已经开发了氧结合新蛋白，包括刚性连接到蛋白主干上的血红素。这缓解了与以前设计中的血红素丢失相关的问题，并允许前所未有地控制新蛋白的特性和功能。由于天然的氧依赖催化需要氧通过受控的电子添加来“激活”，我们将在我们的氧结合新蛋白中探索这一反应，以获得关于能够强大的产氧催化的中间体的生成和稳定性的有价值的信息。最终，我们计划将氧结合和电子传递功能结合成单一蛋白质或具有不同功能的相关蛋白质亚单位的组合。就像模块化家具设计使用腿、抽屉、架子等较小的功能独立子单元的组合并将它们组装成特定的规格一样，我们认为类似的方法可以应用于构建新的蛋白质和酶，其功能由设计师决定。这种方法的一个优点是，通过在人造蛋白质中复制酶和蛋白质的功能，可以从根本上深入了解它们的天然同行的工作原理。

Proteins are biological molecules constructed from linear chains of amino acids that adopt complex 3D structures informed by their amino acid sequence. Each protein typically has a unique structure that is indelibly linked to the function it performs in nature. Enzymes are proteins that catalyze the chemical reactions that occur in the cell, examples of which facilitate the capture and storage of chemical energy from respiration and photosynthesis. The design of new artificial proteins and enzymes remains one of the great challenges in biochemistry, testing our fundamental understanding of the nature of protein as a material. Unlocking the exceptionally powerful array of chemistries that natural enzymes perform promises routes to new drugs, therapies and sources of renewable green energy. Most attempts to construct new enzymes have focussed on modifying natural proteins and enzymes to introduce new catalytic function with modest degrees of success. The problems associated with redesigning natural proteins are due to the layers of complexity that nature incorporates through natural selection into a protein's complicated 3D structure. This complexity serves to complicate functional deconstruction of naturally evolved proteins and enzymes, rendering their redesign intrinsically difficult. We believe that this complexity is not a necessary feature of proteins and enzymes. Our method to effectively avoid such complexity is to work with proteins that have been untouched by natural selection. These simple proteins, neoproteins, are small, robust protein scaffolds with generic amino acid sequences that serve as templates onto which natural protein functions can be added. Non-protein components of certain proteins and enzymes, such as the heme molecule of the protein hemoglobin, can be effectively supported in neoproteins and the various functions that these molecules perform in natural proteins can be exploited. An example of how this method can be effectively used is the creation of a heme-binding neoprotein capable of reversibly binding oxygen, a function common to myoglobin, hemoglobin and the recently discovered neuroglobin. Functional elements of engineering are added step-by-step and the requirements to form such a protein are surprisingly few in number. And, as E. coli produces the artificial protein in large quantities, the oxygen-binding neoprotein is exceptionally cheap to produce and easy to alter through standard molecular biology techniques. Since the oxygen bound state in heme proteins is a pre-requisite for a multitude of catalytic processes in natural proteins, we plan to take inspiration from nature to further the development of these proteins into artificial enzymes. We have developed the oxygen-binding neoprotein to include hemes rigidly attached to the protein backbone. This alleviates problems associated with heme loss from previous designs and allows for an unprecedented control of neoprotein properties and function. Since natural oxygen-dependent catalysis requires that oxygen be 'activated' by the controlled addition of electrons, we will explore this reaction in our oxygen binding neoproteins, gaining valuable information about the generation and stability of intermediates capable of powerful oxygenic catalysis. Ultimately, we plan to combine the oxygen binding and electron delivery functions into either a single protein or a combination of associated protein subunits with discrete functions. Much as modular furniture design uses combinations of smaller functionally independent subunits such as legs, drawers, shelves and assembles them to particular specifications, we think an analogous approach can be applied to the construction of new proteins and enzymes whose functions are dictated by the designer. An advantage of this approach is that through the reproduction of enzyme and protein function in artificial proteins a deep fundamental understanding of the workings of their natural counterparts is gained.